Exosomes‐Loaded Electroconductive Hydrogel Synergistically Promotes Tissue Repair after Spinal Cord Injury via Immunoregulation and Enhancement of Myelinated Axon Growth

Abstract Electroconductive hydrogels are very attractive candidates for accelerated spinal cord injury (SCI) repair because they match the electrical and mechanical properties of neural tissue. However, electroconductive hydrogel implantation can potentially aggravate inflammation, and hinder its repair efficacy. Bone marrow stem cell‐derived exosomes (BMSC‐exosomes) have shown immunomodulatory and tissue regeneration effects, therefore, neural tissue‐like electroconductive hydrogels loaded with BMSC‐exosomes are developed for the synergistic treatment of SCI. These exosomes‐loaded electroconductive hydrogels modulate microglial M2 polarization via the NF‐κB pathway, and synergistically enhance neuronal and oligodendrocyte differentiation of neural stem cells (NSCs) while inhibiting astrocyte differentiation, and also increase axon outgrowth via the PTEN/PI3K/AKT/mTOR pathway. Furthermore, exosomes combined electroconductive hydrogels significantly decrease the number of CD68‐positive microglia, enhance local NSCs recruitment, and promote neuronal and axonal regeneration, resulting in significant functional recovery at the early stage in an SCI mouse model. Hence, the findings of this study demonstrate that the combination of electroconductive hydrogels and BMSC‐exosomes is a promising therapeutic strategy for SCI repair.

Figure S10 Spinal cord pathology after SCI at 3 and 6 weeks. Table S1. Serum and cellular contaminants were negative in the BMSC-derived exosomes detected by the proteomic mass spectrometry. Table S2. Primer sequences of each gene was shown below. Table S3. Sequences of three siRNAs were shown below.   properties of all samples were tested by dynamic oscillatory frequency sweep measurement that showed storage moduli (elastic modulus, G′) of all hydrogels were larger than the loss moduli (viscous modulus, G″) over an angular frequency range of 1-100 Hz, indicating that the hydrogels had good stability. (G) Graph of the quantification of GM, GMP, and GMPE hydrogels mechanical properties (n=5).
(H) Equilibrium swelling properties of GM, GMP and GMPE hydrogels (n=5). (I) GMPE hydrogels exhibited mechanical stability 14 days after soaking in physiological medium (n=5). (J) GMPE hydrogels also exhibited swelling ratio stability 14 days after soaking in physiological medium (n=5) (K) Mouse spinal cord tissue could stick to the GMPE hydrogels in vitro. (L) Force/width-extension curves of the peeling adhesion test. (M) The CV curve of GMPE hydrogel showed its stable electrochemistry 14 days after soaking in physiological medium. (N) 3D IF imaging of hydrogel loaded without exosomes labeled with PKH26 showed that almost no fluorescent PKH26 dye was residual after ultracentrifugation. Statistical differences were determined using an ANOVA with Bonferroni's multiple comparison test (* p<0.05, ** p<0.01, ***p<0.001).

Figure S3 In vitro and in vivo exosomes release from loaded hydrogels. (A) IF image
showing that exosomes can be detected 14 days after they were immobilized in the hydrogel. Scale bars, 50 μm. (B) Exosomes were continuously released into the medium supernatant from the GMPE hydrogel after 14 days (n=5). (C) More than 90% of the exosomes were released from the hydrogels over time (n=5). (D) In vivo bioluminescence imaging showed that when exosomes placed into a mouse model with hydrogel, the hydrogel significantly improves the exosomes retention at the injury site 5 days after implantation. Heatmap scale indicates μW/cm 2 . (E) Labeled exosomes can be detected 5 days after implantation at lesion sites and endogenous cells can phagocytose exosomes released from the GMPE hydrogel. White arrows indicate exosomes taken up by the endogenous cells. Scale bars, 500 μm

Figure S4 Data retrieval, extraction and analysis of transcriptomic miRNAs expression in BMSC-derived exosomes from the Gene Expression Omnibus (GEO) database. (A)
Heatmap of top 100 miRNAs of three different series GSE181530, GSE164965, and GSE119790. (B) heatmap of selective miRNAs of three different series GSE181530, GSE164965 and GSE119790.

Figure S5 In vitro biocompatibility of hydrogels. (A)
A live/dead assay in NSCs was used to evaluate the in vitro biocompatibility of hydrogels. Scale bars, 100 μm. (B) CCK-8 analysis of NSCs was also used to evaluate the in vitro biocompatibility of hydrogels (n=3). The proliferation of cells cultured on the GMPE hydrogel was similar to that on GM hydrogel, and the proliferation rate on GMPE hydrogel was significantly higher than that on GMP hydrogel (n=3). (C) Cytoskeleton staining in NSCs was also used to evaluate hydrogel adhesion. Scale bars, 100 μm. (D) The length of synaptic length was quantified using Image J software (n=11). Statistical differences were determined using an ANOVA with Bonferroni's multiple comparison test (GMPE compared to GMP * p<0.05, ** p<0.01, *** p<0.001; GMPE compared to GM, + p<0.05, ++ p<0.01, +++ p<0.001).

Figure S6 In vivo biodegradability of implanted hydrogels. (A)
The degradation rate of GMPE and GMP hydrogels was significantly slower than that of the GM hydrogel after subcutaneous implantation. (B) HE staining shows the degradation process and inflammation of each hydrogel after implantation. Green arrows indicate where cells have phagocytosed PPy nanoparticles. Scale bars, 50 μm. (C) The thickness of the fibrotic capsule was qualified at 1, 3 and 6 weeks post-implantation (n=3). (D) Quantification of invasive inflammatory cells at 1-, 3-, and 6-week time points (n=3). Statistical differences were determined using an ANOVA with Bonferroni's multiple comparison test (* p<0.05, ** p<0.01, ***p<0.001).

Figure S7
In vivo biocompatibility of implanted hydrogels. (A) HE staining indicating normal morphology in the heart, liver, spleen, lung, and kidney tissues from each treatment group. Scale bars, 500 μm. (B) The level of ALT, AST, TP in serum were similar in all treatment groups, indicating hydrogels did not cause systemic toxicity (n=3). (C) Photograph of serum extracted from whole blood co-cultured with each hydrogel. Samples were light yellow in color and similar to that of the PBS control group, while the Triton-100X group was bright red, indicating its hematolysis. (D) The serum OD values in GM, GMP and GMPE groups were similar to the value in the PBS group, while all were significantly lower than that of the Triton-100X group (n=3). Statistical differences were determined using an ANOVA with Bonferroni's multiple comparison test (* p<0.05, ** p<0.01, ***p<0.001).

Figure S10
Spinal cord pathology after SCI at 3 and 6 weeks. (A) Conventional MRI was used to assess changed in spinal cord pathology in sham and hydrogel implantation after SCI. Write arrows indicate the SCI site. The sites of spinal cord in the transverse section were marked in red dotted frame. (B) Quantitative analysis of lesion volume (n=3) at 3-and 6weeks. Statistical differences were determined using an ANOVA with Bonferroni's multiple comparison test (* p<0.05, ** p<0.01, ***p<0.001).  Table S2. Primer sequences of each gene was showed below. Table S3. Sequences of three siRNAs were showed below.

Target
Sense Antisense